Viral infections pose challenges for effective treatment. While an antiviral treatment may appear to treat the initial acute infection, physical symptoms of infection often return later as persistent infections. A common characteristic of persistent infections is the virus' ability to successfully modulate the immune response to avoid specific and non-specific immune defenses. In essence, persistent viral infections are immunosuppressive diseases. In general, the course of these diseases is moderated by the strength of the immune system. Persistent viral infections are also highly correlated with the development of cancer.
HIV-1 is an example of a virus that causes persistent infections. HIV is perhaps the most widely known of the viruses that cause immunosuppressive diseases. Although the immune system effectively produces antibodies against these viruses in the acute stage of the infection, the antibodies are largely non-neutralizing and allow the infection to progress to the chronic and eventually fatal stages. Moreover, the cytolytic components of the immune system fail to destroy infected cells even though the cells express pathogen-induced cell-surface antigens [1]. The primary therapy against such infections is daily administration of a combination of anti-retroviral drugs that inhibit viral replication after entry into the cell and subsequent maturation. The most commonly used are nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, integrase inhibitors, and protease inhibitors that block enzymatic processing of viral products. These drugs effectively inhibit replication of the virus inside an infected cell and reduce viral load in the blood to undetectable levels [2,3].
A particularly confounding aspect of infections by these viruses is the establishment of latent reservoirs in which the integrated provirus stage can remain dormant for long periods of time. Consequently, the virus cannot be completely cleared from an infected individual by current treatments. Upon discontinuation of anti-retroviral treatment, these reservoirs are activated and the virus “rebounds” to pretreatment levels within a few weeks [3,4]. The question of whether the provirus is indeed dormant or simply replicates at a very low level has not been completely resolved. Often the immune system maintains suppression of viral replication but fails to maintain health when the immune system is compromised.
A defining characteristic of acquired immunodeficiency syndrome (AIDS) is the development of Kaposi sarcoma (KS), a type of cancer that affects cells lining the lymph and blood vessels. KS is caused by herpes virus in immunocompromised subjects.
Like HIV, cytomegalovirus (CMV) cause persistent infections associated with the immune system. CMV, however, is dormant in health individuals and generally becomes active when the immune system is compromised. The antiviral drug ganciclovir, a viral DNA polymerase inhibitor, is commonly used to treat acute cytomegalovirus (CMV) infections. Human CMV has also been found to play a role in the development of cancer through oncomodulation, e.g., enabling cancer cells to evade immune recognitions [44].
Chronic viral hepatitis is the most common risk factor worldwide for liver cancer. Like HIV, Hepatitis C virus (HCV) is a RNA virus and is more likely to result in chronic infection than hepatitis B virus (HBV). Recent advances in the treatment of HCV involve development of protease inhibitors that act in a similar manner as those used to treat HIV-1 infections [5]. For HCV, the protease inhibitors are added to the currently accepted drug regime of pegylated interferon-alpha and ribavirin.
About one-third of the world's population has evidence of a hepatitis B infection, either current or past, which is more than HIV and HCV infections combined [6]. Most healthy adults raise an effective immune response against hepatitis B virus (HBV), but the effectiveness of the immune defense is dependent upon the activity of natural killer (NK) cells [6]. NK T cells contribute to resolution of a HBV infection, with the NKG2D receptor playing a key role [6]. HBV establishes a chronic infection, and although infected cells express the hepatitis B surface antigen (HBsAg), the immune system is unable to prevent progression of the infection. Long term continuing virus replication leads to progression to cirrhosis and hepatocellular carcinoma [6-8]. Infection by HBV is the leading cause of hepatocellular carcinoma. Approximately 662,000 deaths occur worldwide each year, with roughly half of them in China [9].
A potentially powerful therapeutic approach for these persistent viral infections and immunosuppressive diseases is a combination of drugs with antiviral activity, in particular those that bind to NKG2D, and those with strong anticancer activity that promote induction of proliferation of activated cells of the innate and adaptive immune system.
An Alternative Approach to Therapy
In contrast to therapeutic approaches aimed at prevention or control of disease by directly inhibiting a step in the viral replication cycle, as described above, or by the use of highly toxic cytotoxic chemotherapeutic drugs for cancer treatment, reactivation of patients' immune system is an alternative therapy that holds promise for restoring health and productivity to an infected patient in a practical, cost-effective manner. This approach provides a general defense against diseases rather than a pathogen-specific treatment. As a result, an intense interest in immunotherapy, as indicated by the development of cytokine and monoclonal antibody treatments, is leading to products that can stimulate or inhibit the immune system.
The role of cytokines in the inhibition of HIV infectivity, particularly interleukin-16 (IL-16), interleukin-8 (IL-8) and RANTES (Regulated upon Activation, Normal T-cell Expressed, and Secreted; also known as CCL5), is very important. Cytokines such as IL-16, IL-8 and RANTES, which have overlapping and complementary functions, can act to attenuate viral infection by competing with viral binding with the receptors and by interfering with viral entry into cells by down-regulating the receptors required for entry. Other cytokines such as interferons (e.g., IFN-α. and IFN-γ) act to reduce viral load by activating intracellular anti-viral enzymes and also by stimulating antibody-mediated phagocytosis. These cytokines have also been shown to be effective in the acute stage of HBV infection [6].
Interleukins (IL's) and interferons (IFN's) are potent cellular stimulants that are released from a variety of cells in response to insult or injury. Consequently, these proteins have attracted intense interest as therapeutic agents. IL-16 is a natural ligand of CD4 and should compete with virus for binding to T cells. IL-21 is required to avoid depletion of CD8+ T cells and also essential to maintain immunity and resolve persistent viral infections [10-12]. Similar to general stimulants such as lipopolysaccharide (LPS), however, IL's and IFN's induce release of inflammatory cytokines. Therefore, when given at higher than normal endogenous concentrations, they often have substantial adverse effects, which can be life threatening and may require inpatient treatment facilities. Similarly, levels of TNF-α, IL-1β and IL-6 are directly correlated with the probability of death in humans. Moreover, production of recombinant IL's and IFN's and their application are very costly. Even lower-dosage immunostimulant treatments developed for out-patient use have lower success rates and are not suitable in some situations such as, for example, to extend remission from cancer therapy or control a disease such as HIV at a chronic level. In view of this, it appears that exogenous therapeutic agents such as large, intact cytokine molecules are not well suited for general therapeutic use.
Usually, infections are cleared by the immune system through (i) internalization of the pathogen and presentation of antigens to T and B cells by dendritic cells (DCs), (ii) generation of antibodies by B cells, (iii) lysis of pathogen-infected cells by NK cells and CD8+ cytotoxic T lymphocytes (CTL), and/or (iv) destruction of the virus or cancer cell by antibody-mediated phagocytosis. While neutralizing antibody responses are subject to pathogen escape, many non-neutralizing antibodies that nevertheless bind the pathogen are present in infected patients. Restoration of immune effector cell functions, in particular phagocytic activity, which can recognize the resulting antigen-antibody complexes and destroy the complexes by antibody (Fc)-mediated phagocytosis, may be applicable to the clearance of infections in general.
The cell types that have significant involvement in viral infections in addition to phagocytic cells are in particular, two subsets of the T cell population (CD3+ and CD8+), NK cells (CD56+) and CTLs (CD8+). These cells are able to kill virus-infected cells and cancer cells by antibody-dependent cellular cytotoxicity (ADCC) in addition to an ability to directly lyse infected cells. NK cells are an integral component of the innate immune system and are primarily responsible for killing virus-infected and cancer cells. NK cells and CTL kill their targets mainly by releasing cytotoxic molecules such as perforin, granzymes and granlysin, which are contained in intracellular granules. These molecules are released when these cells make contact with target cells that contain antigens on the surface of viral infected or cancer cells to which antibodies bind. Activated NK cells also release cytokines and chemokines such as IFN-γ that activates macrophages and drives differentiation of CD4+ T cells into type 1 (Th1) cells [11,12].
Information relevant to attempts to address one or more of these problems can be found in the following references: U.S. Patent Publication No. 2007/0003542; U.S. Patent Publication No. 2006/0269519; U.S. Patent Publication No. 2004/0248192; P. W. Latham, 1999; Fatkenheuer et al., 2005; Stover et al., 2006; Cohen, 2007; GlaxoSmithKline, 2005a and GlaxoSmithKline, 2005b. Each one of these treatments referred to in these references, however, suffers from one or more of the following disadvantages:
1. the size or composition of the agent provides significant challenges to cost-effective synthesis and purification;
2. the agent is specific for particular pathogen and/or cell type, rendering them unsuitable for general therapeutic use;
3. treatment with the agent induces clinically deleterious side effects that can be life-threatening, such as inflammation or hepatotoxicity, and require inpatient treatment facilities;
4. termination of treatment is followed soon thereafter by an increased systemic viral load;
5. long term exposure to agent often leads to treatment-resistant pathogens;
6. lower-dosage treatments developed for out-patient use have lower success rates and are not suitable in some situations;
7. treatment is ineffective, impractical, or cost-prohibitive for a large proportion of patients;
8. development of therapeutic antibodies require considerable medical infrastructure;
9. treatment such as vaccines may be appropriate to prevent infection but not to treat those already infected and who have a suppressed immune system;
10. no beneficial synergy between the immunogenic response induced and the effects of other endogenous immunoregulators;
11. agent inhibits the release of inhibitory cytokines that suppress release of beneficial cytokines, an indirect treatment; and
12. agent acts to restore baseline cytokine levels to balance responses of the immune system rather than promoting activation of phagocytes.
Many of these therapeutic protocols also become ineffective with time because mutation of the pathogen allows it to escape the treatment. Moreover, any immunosuppression that accompanies the disease attenuates the ability of the innate and adaptive immune systems to respond to antigenic changes and thereby keep the infection under control.
The immune system in individuals infected with a pathogenic agents such as HIV-1 or HBV initiate a defense response by production of antibodies. Even though the virus may mutate at one or a few sites and thereby escape the neutralizing activity of antibodies, endogenously produced non-neutralizing antibodies are usually polyclonal and may still bind the virus. The presence of anti-viral antibodies is often used as a diagnostic test for infection. During the course of the disease, the cellular components of the innate and adaptive immune response then become absent or quiescent. When the immune defense mechanisms reach a sufficiently low level, viral replication is not held in check and rapidly leads to a final stage of the disease. However, even at this late stage, patients can be rescued from death by aggressive therapy. Therefore, an agent that reactivates cells of the immune system, in particular phagocytes and NK cells, will likely restore an immune defense against progression of the disease.
Not only is it essential to overcome the suppressive mechanisms of the pathogen, it is also important to modulate the host's natural mechanism that suppress the immune system. Therapeutic agents that activate/reactivate the immune system show particular promise in this regard, including cytokines and immunomodulators, although therapies based on exogenous agents such as large, intact cytokine molecules are not generally well suited for therapeutic use. Peptides, however, are often much more suitable therapeutic agents than large polypeptides or proteins. Peptides can, for example, be designed to induce one or more particular desired effects in vitro or in vivo, often without concomitantly inducing deleterious effects, and can usually be synthesized in a cost effective manner.
The development of this technology is applicable to diseases caused by viruses, bacteria, fungi and cancers.